Respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory illness and hospitalization in young children and has long been recognized as a priority disease for a vaccine. RSV presents many challenges for vaccine efficacy. Over the nearly 40 years since the failed formalin-inactivated RSV vaccine great strides have been made in understanding the appropriate immune responses for protection versus immunopotentiation. Vaccine-evoked protection against RSV-induced disease of the lower respiratory system must include antibody and CD8+ cytolytic T- cells. Of equal or greater importance, the vaccine must not stimulate interleukin (IL)-4 and IL-5, indicators of a TH2 CD4+ response. IL-4 has been shown to immunopotentiate while the TH1-associated cytokine interferon-gamma and CD8+ T-cells have been correlated with protection without enhanced lung pathology. Thus, we propose that vaccination will be effective if there is induction of balanced helper and killer T-cell responses to a key RSV antigen. Adenovirus vectors (advectors) have shown promise as vaccines for infectious diseases. Preclinical and clinical testing of replication-defective advector vaccines with potent expression of the encoded antigen is underway for HIV, Ebola, Influenza, and other diseases. Advectors are capable of stimulating Th1 helper and cytolytic CD8+ responses and neutralizing antibody to the vectored antigen. The overall goal of this SBIR application is to test whether immunization with novel advector vaccine candidates for RSV stimulate neutralizing antibody and virus-specific Th1 CD4+ and CD8+ T-cells without Th2 CD4+. We will build novel recombinant adenovirus vectors that express the RSV F protein to high levels. GenVec has cell lines to construct advectors based on different serotypes of adenovirus, which provides advectors that can be used in prime-boost regimens and circumvent pre-existing anti-vector immunity. These same cell lines facilitate the construction of advectors that express transgenes that are cytotoxic or inhibit advector replication. Additionally, GenVec's cell line has been used as the cell substrate for the generation and manufacturing of an advector-based HIV vaccine that is now in phase II clinical testing. The advectors will be tested for the pattern of immune response evoked to RSV, for efficacy against RSV challenge, and for lung histopathology. Both cotton rat and murine models will be used to fully characterize RSV protection, safety, and the immune response. From the phase I portion of this proposal key immunological and safety data will be generated to enable decision-making for advancement to phase II. In the phase II portion, lead adenovectors will be chosen for additional preclinical efficacy testing, preclinical safety testing, and manufacturing in preparation for clinical testing. PUBLIC HEALTH RELEVANCE: Respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory illness and hospitalization in young children and has long been recognized as a priority disease for a vaccine. We propose a method of vaccination that will induce an antibody and balanced helper and killer T-cell response against RSV without causing enhanced disease. In phase I we will generate novel replication-deficient adenovirus vectors that express F protein and test them for safety, generation of appropriate immune responses, and protection against RSV.